Project Summary: While traditionally conceived as passive support elements for neuronal networks, non-neuronal brain cells are now appreciated as dynamic integral components of central nervous system (CNS) circuitry. Astrocytes, for example, serve as powerful regulators of neuronal spiking, synaptic plasticity, and brain blood flow. Similarly, microglia not only respond to a wide range of CNS perturbations, but also participate in circuit development and plasticity through the active elimination of synaptic connections. Dynamic oligodendrocyte and brain endothelial cell responses to neuronal signaling also play key roles in shaping and homeostatically stabilizing neuronal circuit activity. While dysfunction of these glial and vascular cell types is implicated in a wide range of neurological disorders, study of the diversity and function of glial and vascular cells has suffered from an historical underinvestment in basic tool development relative to that focused on neuronal cell types. In this regard, one major persistent impediment to ongoing investigations stems from our limited ability to genetically access specific glial or vascular cell types, with many of the existing reagents displaying significant shortcomings. Capitalizing on recent advances in epigenomic analysis and single-cell profiling, we recently developed and validated a scalable strategy for the generation of cell-type-specific adeno-associated viral (AAV) drivers incorporating cell-type-restricted gene regulatory elements (GREs). Here we propose to apply this novel approach to isolate viral drivers that are specific for four distinct classes of non-neuronal cell types: astrocytes, oligodendrocytes, microglia, and endothelial cells. Moreover, these viral libraries will be designed to enrich for candidate drivers specific for distinct fine-grained subtypes within each of these broad cell type classifications, including subtypes not specifically accessible with existing transgenic lines. To this end, we propose the following specific aims: 1) Single-cell profiling of AAV serotype tropism; 2) High-throughput screening for cell-type-restricted GRE-driven AAV reporters; and 3) Validation and in vivo characterization of newly isolated GRE-driven cell-type-restricted AAVs. These studies should significantly expand genetic access to a broad array of functionally relevant non-neuronal brain cell types. The viral vectors developed in this study will possess immediate utility for a variety of cell-type-specific applications. Furthermore, given that previous viral drivers have been found to largely retain their specificity in other species, this strategy should provide important new tools for future investigations in additional experimental contexts, particularly in genetically inaccessible model organisms such as primates. !